Most immune responses involve many components of the immune system. Although the immune mechanisms involved in the elimination of malignantly transformed cells are not well understood, it is reasonable to assume that if more immune mechanisms are activated and enhanced, the tumorous cells may be eliminated more effectively. Also, both humoral and cellular mechanisms are known to be involved in the immune response against viruses and virus-infected cells. Thus, generally speaking, for the therapeutic treatments of patients with various cancers or infectious diseases, and for protecting individuals exposed to infectious agents from contracting the infection, it is desirable to enhance the entire immune system.
The various branches of the immune system include antibodies, cytotoxic T cells (CTLs), T cells mediating delayed-type hypersensitivities (T.sub.TDH cells), monocytes and macrophages, natural killer (NK) cells, K cells mediating antibody dependent cellular cytotoxicity ("ADCC"), and granulocytes. Complex interactions are involved in the activation of these various branches. The helper T cells (T.sub.h cells) play central regulatory roles, and many factors are secreted by these cells and other cells in a certain concerted fashion during the activation and proliferation phases. There is good reason to believe that the concerted production of lymphokines and cytokines, at the appropriate time and in the proper relative proportions, is important for maximizing the immune response.
Potentiation of the immune system is desirable for treating a number of pathologic conditions, e.g., for treatment of malignant tumors. The immune potentiators include substances identified from screening natural sources, such as cultures of microorganisms, marine animals, herbs, or plants, as well as substances screened from large batteries of synthetic organic compounds.
One example of a substance from a natural source is muramyl dipeptide, which has been identified as the smallest structure from the cell wall of staphylococcal bacteria which still retains immune potentiating effects. Many analogues of muramyl dipeptide have been synthesized. Muramyl dipeptide and its analogues are macrophage activators, and have been tested and developed as therapeutic agents for tumors and as adjuvants for vaccines.
Other examples of immune potentiators derived from natural sources include double-stranded RNA and mismatched double-stranded RNA (also called Ampligen) which can induce interferon synthesis and other immune functions. These substances have also been tested for treating tumors and viral diseases, such as AIDS.
Immune potentiators may be applied to patients alone or in combination with surgery, irradiation, or chemotherapy. They may also be desirable for treating patients with viral infectious diseases or for protecting individuals, after exposure to viruses, from contracting infection. Immune potentiators may be useful as adjuvants for various vaccines for infectious diseases or cancers.
Recently, recombinant human lymphokines and cytokines have been produced by genetic engineering. Many such recombinant "biological response modifiers" are being tested for treatment of various cancers and infectious diseases. A few recombinant products, such as interleukin-2 (IL-2), .alpha.-interferon, .gamma.-interferon, granulocyte-colony stimulation factor and granulocyte/monocyte-colony stimulation factor (G-CSF, GM-CSF), have been approved in many countries for use against certain cancers and infectious diseases. For example, IL-2 is approved for treating patients with renal cell carcinoma; .alpha.-interferon is approved for treating patients with hairy cell carcinoma or with hepatitis B infection; G-CSF and GM-CSF are approved for treating cancer patients receiving chemotherapy for the purposes of restoring lost neutrophils.
Individual recombinant lympholines, such as IL-2, IL-4, or .gamma.-interferon can augment some aspects of the immune system, but function only against limited immunocyte targets and can only potentiate certain immune functions and not the entire immune system. They also probably function only over short ranges and in limited areas in vivo. Also, cytolines and lymphokines which are injected into patients are cleared rapidly through the kidneys. They likely will not be present in sufficiently high concentrations in the lymphoid system for long enough to achieve their desired immunological effects.
Of the various substances other than lymphokines or cytokines which have been studied for potentiating the immune system, most are suitable for in vivo use do not target or enhance the T cells directly. For example, muramyl dipeptide, and analogues thereof, primarily activate macrophages. Double-stranded RNA and mismatched double-stranded RNA mainly induce interferon production by a variety of cells.
A few naturally-derived protein substances are known to be potent T cell mitogens in culture in vitro, and have been used in studies to characterize and quantitate T cell activity. These substances include phytohemagglutinin A (PHA), concanavalin A (Con A), wheat germ agglutinin (WGA), and some other lectins, defined as carbohydrate-binding plant proteins. However, these T-cell mitogenic proteins, although very useful for in vitro studies, have poor specificity and therefore bind to almost all cell types. Because they are toxic and lack specificity, they are not effective for in vivo use as T cell potentiators.
In order to activate and expand lymphocytes to achieve satisfactory therapeutic effects while avoiding administering toxic substances, some groups have sought to activate and expand the T lymphocytes from patients in culture in vitro for a period of time under optimal conditions and then harvest the activated cells and inject them back into the same patients. In this so-called IL-2/LAK therapeutic regimen, used by the Biological Therapy Institute (Franklin, Tenn.) to treat patients with various cancers, the blood is first drawn from the patients and the mononuclear cells are isolated. See Rosenberg, S. A. et al., N. Eng. J. Med. 316:889 (1987). The cells are incubated in medium containing recombinant IL-2 for several weeks, and the activated and expanded T cells, which contain the lympholine-activated killer (LAK) cells, are harvested and injected into the patients.
A more recent, modified version of this IL-2/LAK therapy, known as autolymphocyte therapy (ALT) has been developed by Cellcor Therapies, Inc. in Boston, Mass. See Osband, M. E. et al., Lancet 335:994 (1990). The lymphocytes from renal cell carcinoma patients are taken six times. The first time, the lymphocytes are stimulated with antibodies specific for human CD3 antigen (anti-CD3) in vitro to produce lymphokines. The culture supernatant is collected after a few days of culturing, and the cells are discarded. Then, the second through the sixth time, the lymphocytes taken from the patients are incubated in the "autologous" lymphokines for a period of five days and the cells are harvested and injected into the same patients.
It is claimed that these approaches, involving in vitro lymphocyte stimulation and expansion, achieve beneficial responses in a portion of the treated patients. The major concern with these regimens is that the treatment is very tedious, expensive, and requires a sophisticated, specialized cell culture facility. The variation among cells or cultures from different patients requires demanding monitoring procedures. Also, lymphocyte cultures have very poor viability even under optimal conditions, meaning that during the culturing, large numbers of the cells will die. When large numbers of dead cells are injected into patients, this may actually burden the reticuloendothelial system (RES) and reduce its effectiveness in combating the tumor cells.
In summary, the clinical studies and approved routine uses of IL-2 and .gamma.-interferon and of LAK or ALT therapies indicate that T cell activation and expansion can achieve therapeutic effects in some patients with cancers or infectious diseases. On the other hand, the results of these treatments suggest that the lymphokine treatments have certain deficiencies and the LAK and ALT treatments have some substantial drawbacks. An efficacious and feasible treatment may be achieved if these deficiencies can be eliminated.
A number of MAbs specific for CD3 on the surface of human T cells (pan T marker) are known to be very potent mitogens of human T cells in vitro, e.g., the MAb OKT3. Van Wauwe, J. P. et al., J. Immunology 124:2708 (1980); Chang, T. W. et al., Proc. Natl. Acad. Sci. U.S.A. 78:1805 (1981); MAb 64.1 Hansen, J. A. et al., Leukocyte Typing: Human Leukocyte Differentiation Antigens Detected by Monoclonal Antibodies, Eds. Bernard, A. et al. (Spring Verlag, N.Y., 1984). In medium containing only fetal calf serum and no human serum (and therefore no IgG), the anti-CD3 MAbs are much more potent than PHA or Con A in inducing T cell proliferation.
But the mitogenic effect of anti-CD3 requires both specific binding to the CD3 antigen and the presence of the Fc moiety of the antibody, as well as the presence of monocytes and macrophages. The best explanation for these results is that the Fc of the anti-CD3 MAbs binds to the Fc receptors on monocytes/macrophages, thereby aggregating the CD3 antigen on the T cell surface. Since CD3 is associated with the T cell antigen receptors, the aggregation of CD3 triggers the activation and proliferation of the T cells.
This explanation is supported by experiments which show that when the anti-human CD3 MAb is conjugated to Sepharose 4B beads or coated on the substratum plastic surface of culture wells, monocytes and macrophages are not needed to induce activation and proliferation of T cells. See Williams, J. M. et al., J. Immunol. 135:2249 (1985); Ceuppens, J. L. & Baroja, M. L., J. Immunol. 137:1816 (1986); Geppert, T. D. & Lipsky P. E., J. Immunol. 138:1660 (1987). Based on these experiments, it has been suggested that the solid-phase anti-CD3 MAb functions by aggregating the CD3 antigen on the T cell surface.
However, when anti-human CD3 is injected in vivo, the results are the opposite of the in vitro effects. OKT3 MAb, which is the first MAb ever approved for therapeutic use in vivo, is strongly immunosuppressive and is approved for use as an immunosuppressor for patients receiving kidney transplants. Ortho Multicenter Group Study, N. Eng. J. Med. 313:337 (1985). The injection of OKT3 causes rapid depletion of T cells from the circulation.
Administration of anti-CD3 monoclonal antibodies (or F(ab').sub.2 fragments thereof) to mice also causes T cell depletion. Hirsch, R. et al., TRANSPLANTATION 49:1117-23 (1990). This monoclonal antibody also caused weight loss, diarrhea, and decreased activity within 24 hours in all mice, and death of one-half of the mice it was administered to in 3 days. Hirsch, R. et al., id. at p. 1118, col. 1, paragraph 2. In another study it was observed that mice administered 40 .mu.g of this anti-CD3 antibody seemed to develop more rapidly growing tumors. Ellenhorn, J. D. et al., Science 242:569-571 (1988) at p. 570, in explanation of FIG. 3. These observations are consistent with the observation that this antibody caused depletion of T cells.
Although the mechanism by which anti-CD3 causes this rapid depletion of T cells is not well understood, the best explanation is that anti-CD3 induces ADCC of the T cells, i.e., as the T cells coated by anti-CD3 circulate through the spleen and liver, they are lysed by the phagocytic cells of the RES in these organs. It is also possible that some of the T cells are destroyed by complement-mediated cytolysis and some other cytolytic mechanisms. Thus, it is expected that conjugates of the OKT3 MAb with other substances will also cause depletion of T cells in vivo.